Questions tagged [rna-seq]

Questions should include this tag if they pertain to issues related to bioinformatics analysis of RNA-seq data, e.g. normalization, differential expression analysis, sequencing and experimental design or transcriptome assembly...

Filter by
Sorted by
Tagged with
0
votes
2answers
38 views

Identifying nested/overlapping reading frames in nucleotide sequences

I'll start by saying I'm a total stranger to this field, so I'd love to be corrected if I misunderstood something along the way. I'm writing a python code to identify all the reading frames in a ...
1
vote
2answers
29 views

Mapping statistics from bam file using bbtools and sambamba

Below are the statistics for RNA-seq mapped and unmapped paired-end reads to rice genome using reformat.sh from bbtools on bam files. It gives 77% mapped and 5% unmapped, what about the remaining 18% ...
2
votes
1answer
172 views

load the phenotype data for ballgown

I am trying to reproduce the work of this paper [1], and I have run StringTie successfully, but after that I have to run Ballgown but could not understand this command: ...
0
votes
1answer
9 views

Running GSEA or comparable analysis with multiple variable interaction?

I am working with an RNA-seq dataset generated from a complex experimental design. Our main question is focused on the interaction between two variables, one (INJ) with two conditions (L,S) and the ...
0
votes
3answers
94 views

RNAseq biological replicates not clustering in PCA plots

I have RNAseq data from 4 samples with 3 biological replicates per sample. I am currently trying to do the differential expression analysis with DESeq2 but the biological replicates will not cluster ...
1
vote
2answers
84 views

Advantages of paired-end sequencing compared to single end

One of the advantages of paired end sequencing over single end is that it doubles the amount of data. Another supposed advantage is that it leads to more accurate reads because if say Read 1 (see ...
1
vote
4answers
48 views

How can I obtain FTP links to studies in ENA?

How can I programmatically obtain ftp links to RNA seq fastq files in ENA? Here's an example of a link that I would be interested in obtaining: ...
1
vote
1answer
213 views

Downloading SRA Files from AWS

I want to download the original BAM files that the authors had uploaded to SRA. Normally, I would just use sam-dump, but the files are having issues that seem ...
0
votes
1answer
36 views

Setting Contrast for DESeq2 results

This question was also asked on BioStars I am trying to recreate this heatmap. How would I compare the four variables: Ly49+/- and MOG/MOGSP to get a single heatmap? Thank you for helping me through ...
0
votes
1answer
21 views

Pointing Cromwell to local EBS mounted storage on AWS Batch

I'm trying to run the WDL tasks from the GTEx RNA-Seq pipeline with Cromwell using AWS Batch as a backend. I store the STAR alignment index on an Elastic Block Store since my Cromwell server instance ...
0
votes
1answer
45 views

Error using htseq-count: Could not retrieve index file

I have a total RNAseq dataset that I aligned using STAR producing BAM files (sorted by coordinates). I am now trying to get counts for the lncRNA sequences using htseq-count, with the command: ...
0
votes
1answer
61 views

Interpreting this plot from GSEA

I have RNA-seq for two groups of patients: Responders to chemotherapy (n=9) versus ...
2
votes
3answers
89 views

Getting hierarchy of cell populations with Drop-seq data

I plan on collecting some drop-seq data from brain tissue (from mice and humans). Using only the 75-85% of genes that have 1:1 orthology between mice and humans, I'd like to cluster the cells by ...
8
votes
3answers
1k views

Convert R RNA-seq data object to a Python object

I have done some work in R and would like to try a Python tool. What is a good way to import the data (and its annotations etc) as a Python object? I am particularly interested in converting a ...
2
votes
1answer
69 views

How to visualize genome track of gene in specific cell-lines?

I'm trying to make a plot showing genome tracks of specific genes in specific cell-lines of RNA-seq and Chip-seq data. It should look something like this I have recently seen this encode, but in ...
1
vote
1answer
60 views

Cuffmerge: EOF marker is absent. The input is probably truncated

I need to merge my all transcripts.gtf from cufflinks output follow this command line : cuffmerge -o merged_gtf_output -p 15 -s ref.fasta -g anot.gtf assembly.txt ...
0
votes
0answers
27 views

Which of the Transcriptome assembly method is best for identifying novel lncRNAs?

I'm working with human samples and I'm trying to identify novel lncRNAs from tumor samples of Prostate cancer. I'm using reference based transcriptome assembly with ...
0
votes
2answers
39 views

Recommended workflow for RNA long read Kallisto-like TPM estimation?

On short reads, Kallisto/Salmon is a standard workflow for measuring RNA transcript counts in TPM. However, when I tried to Google a similar workflow for long reads, it's not clear there is a ...
0
votes
1answer
24 views

Analyzing transcripts for non-coding RNA

From a RNA seq experiment we look for the counts of transcripts of tRNA but we are also interested of other types of RNA we can find. Because of the protocol used for the library prep we can't analyse ...
0
votes
1answer
37 views

the variation between treatments is less than the variation between replicates in RNA-seq data

I have a set of RNA-seq samples from targeting different proteins in a complex with siRNAs. However, the ...
0
votes
0answers
20 views

How to find siRNA off-targets from RNA-seq data?

I have an RNA-seq dataset from siRNAs targeting different components of a molecular pathway. the problem is that the siRNA ...
0
votes
3answers
485 views

Which correlation method to compute the correlation score between different clusters of Sc-RNAseq data?

I'm analyzing single cell rna-seq data and trying to compute the correlation score between different clusters. Wondering how to choose the correlation method("pearson" (default), "kendall", or "...
0
votes
1answer
40 views

NO_COOR reads not in a single block at the end 0 -1

I am doing an RNA seq analysis with mouse samples. I had my reads in several lanes, so I concatenated those fastq reads. Then I've mapped my reads with Hisat2 against the new version of mouse "...
0
votes
1answer
62 views

How to add cluster name to metadata in Seurat?

I'm working on a Seurat object and want to name the clusters according to 2 values alone (yes/no). So I want to add a new column to metadata and annotate the clusters (UMAP) with it. ...
1
vote
1answer
49 views

TCGA: gene IDs not appearing and other concerns

I want to download RNA-seq datasets of 4 or 5 different types of cancer from the TCGA to investigate what is happening with my gene of interest. The problem is that I'm a physician and I don't know ...
8
votes
4answers
220 views

How can the cell line contribution be estimated from RNASeq data?

Using a laser-capture microdissection of cells a group of cells stained with the marker of interest was sequenced. In another cohort of patients (this is all human liver tissue) the whole tissue was ...
3
votes
1answer
98 views

Removing Batch Effect in Heatmaps after Differential Gene Expression Analysis

I'm working on a dataset in which the first replicate of each group is one batch and the second replicate is in a second batch. After checking the PCA plot and ...
0
votes
1answer
113 views

RNASeq: Normalization, stabilization, gene length and rlog

I was thinking about the best method for normalization, which takes gene length into account (in order to compare genes)... Do you think I can do that? : - taking raw counts and dividing each gene by ...
1
vote
1answer
46 views

Within and between sample count normalization

recently I came across a situation where RNASeq sample quality was expresses trough DESeq sizeFactor. So authors reported quality of their samples with respect to some publicly available datasets as ...
0
votes
2answers
34 views

why in 3' quantseq should we use reference genome and not transcriptome?

I am studying about RNA seq. I have found that in 3'prime rna seq (quantseq, tagseq) we should use a reference genome with good annotation (plus known UTRs). My question is why? Why could not ...
1
vote
1answer
90 views

why in RNA seq don't we only use reference transcriptome?

I would like to ask why in RNA seq analysis (alignment step) we use sometimes reference genome instead of reference transcriptome? thank you!
0
votes
0answers
50 views

Differential Gene Expression with Replicates for some of the samples

[this question has also been posted on Biostars; some additional clarification from there has been copied into this question] I've been asked to analyse a set of samples in which their control sample ...
1
vote
0answers
23 views

What is a good rule of thumb for the threshold of noise versus signal for RPK in RNA seq?

I have RPK values (RNA seq) and I'm wondering what is a good rule of thumb for what is considered to be noise versus what is considered to be signal? I.e what should I choose as a threshold value for ...
0
votes
0answers
13 views

What is the best way to address the question of doublets and multiplets in a single cell RNA seq data set?

I have attached a histogram plot of the number of genes per cell in a single cell RNA seq data set of lung endothelial cells. I do not find a bimodal or multimodal distribution of the number of genes ...
0
votes
0answers
32 views

a proper Design Matrix for several drug treatments with both control negative and control positive

I have a dataset of RNA-seq samples for testing different drugs on the presence of another drug. One of my samples is the normal cells with no drugs (control negative) and another is the cells with ...
0
votes
2answers
42 views

How to get transcriptome FASTA file for viruses for Kallisto pseudo-alignment?

I guess the title is self-explanatory. I'd like to find the reference transcriptome files for a few human viruses. My RNA-seq samples are from human tumor cells that were infected with viruses. The ...
1
vote
0answers
18 views

What is the best approach to classify a patient cohort by the expression (low, int, high) of a single gene of interest?

I am working with a dataset of nearly 100 patients. I performed Salmon quantification with genecodeV34 and imported the results with tximeta. I normalised the TPM salmon output with TPM (using edgeR, ...
1
vote
1answer
21 views

classifying samples by TCGA signature

I have some RNA-seq samples from multiple glioblastoma tumours that I'm now trying to classify according to a specific gene signature (from Verhaak et al., 2010) using R. The gene signature is ...
0
votes
1answer
110 views

CIBERSORT runtime error eval failed

I was running CIBERSORT but caught this error right after it started permutation: ...
0
votes
0answers
20 views

particular heatmap combinning to barplot for up and down-regulated genes

I have a list of genes (regulated up and down) by Deseq2 . I'd like to generate a heatmap of a particular combination for the barplot like in this graph : which I read in this paper: https://www....
0
votes
2answers
62 views

can I download DESeq2 in R 3.6.3 in Linux MInt?

I am trying to download DESeq2 in R 3.6.3. Is it possible? This is printed when I am trying: Warning in install.packages : package ‘DESeq2’ is not available (for R version 3.6.3) Thank you!
1
vote
3answers
74 views

Download multiple fastq files using fastq-dump

I want to download the following fastq files at the same time in Salmon: - SRR10611214 - SRR10611215 - SRR10611215 - SRR10611216 - SRR10611217 Is there a way ...
2
votes
0answers
28 views

scRNA-Seq: Account for sequencing depth and gene length?

Task: Normalize a single-cell RNA-Seq dataset to account for sequencing depth and gene-length. For UMI-count based protocols (like 10x) that don't suffer from gene-length biases, there are various ...
0
votes
0answers
17 views

Removing Ribosomal RNA genes from Drosophila gene counts

Hi I have seen similar questions but I still need clarification on Removing ribosomal RNA genes from a gene counts matrix (Drosophila Melanogaster) Finding a fasta file with only ribosomal genes, I ...
2
votes
1answer
23 views

Comparing multiple treatments to multiple other treatments in edgeR for simple effects in a complex experimental design

I am working with a RNA-seq data set in maize that has a relatively complex design. There are two levels of treatment A (nitrogen fertilizer level in the field, high or low), two levels of treatment B ...
0
votes
0answers
14 views

Detecting Transcript Isoforms with HISAT2/Stringtie RNAseq Output

I have the following Stringtie RNA-seq output files for several samples in the image below. For additional context, I have utilized as part of my RNA-seq workflow. HISAT2, samtools, and Stringtie ...
0
votes
1answer
44 views

about two group comparison of three group data in DESeq2 package

When we have three groups samples (A,B,C) with 25000 genes and the main interest is A vs B, should we limit samples only A and B for normalization to perform DEG analysis? Or better to include all ...
0
votes
1answer
39 views

What is a good RNA seq normalization method that allows for across sample comparisons and between transcripts

What is a good RNA seq normalization method that allows for across sample comparisons, and allows between transcripts comparisons as well? I read that TMM for example allows across sample comparisons ...
0
votes
0answers
14 views

Suggestion for a single-cell analysis: how to orchestrate a single cell analysis in order to infer the cell types?

I'm very new to scRNA-seq data so I'm sorry if I eventually posed a trifling question. I orchestrated a sc-cell experiment passing through several steps. First of all I eliminated all the genes that ...
0
votes
1answer
84 views

Which module to select for Pathway analysis based on Module trait correlation and pvalue?

I have a total of 35 tumor samples classified into 4 subtypes. Subtype A, B, C, and D. I have RNAseq data. I'm interested in identifying modules related to each subtype with co-expression network ...

1
2 3 4 5
9